The present invention concerns a suspension of crystallized particles of an EPI-hNE protein, methods for preparing said suspension, a dry powder aerosol derived from said suspension, an inhalable pharmaceutical formulation comprising said suspension or said dry powder aerosol, and the use of said inhalable pharmaceutical formulation in the treatment of various pathological conditions.
International Patent Application WO 96/20278 to Ley et al. describes a number of genetically engineered novel proteins which inhibit human neutrophil elastase (hNE). As indicated in the above-cited patent application, human neutrophil elastase (also known as human leukocyte elastase) is one of the major neutral proteases of the azurophil granules of polymorphonuclear leukocytes. This enzyme is involved in the elimination of pathogens, and in connective tissue restructuring.
The principal systemic inhibitor of hNE is the xcex1-1-protease inhibitor, formerly known as xcex11 antitrypsin. In a certain number of pathological situations (hereditary disorders, chronic bronchitis, emphysema, cystic fibrosis), this inhibitor is either not present in sufficient amounts in the bloodstream or is inactivated, leading to uncontrolled elastolytic activity of hNE, which causes extensive destruction of lung tissue.
WO 96/20278 thus proposes novel proteins which are stable, non-toxic, highly efficacious inhibitors of hNE. These inhibitors form part of a group of inhibitors derived from a Kunitz-type inhibitory domain found in basic pancreatic trypsin inhibitor (BPTI) or a protein of human origin, namely the light chain of human Inter-xcex1-trypsin inhibitor (ITI). They are, inter alia, EPI-hNE-1, EPI-hNE-2, EPI-hNE-3 and EPI-hNE-4. The inhibitors of WO 96/20278 are produced by biotechnological methods and contain modified DNA sequences, with respect to the biological Kunitz domains, which render them highly potent. One of these inhibitors, EPI-hNE-4, is of particular interest.
WO 96/20278 describes preparation of Pichia pastoris production systems for hNE inhibitors EPI-hNE1, EPI-hNE-2, EPI-hNE-3 and EPI-hNE-4, protein production and purification (see in particular Examples 10-15).
Yeast Pichia pastoris mutant strain GS115 containing a non functional histidinol dehydrogenase gene (his4) was transformed by expression plasmids comprising a sequence encoding the S. cerevisiae mating factor alpha prepro peptide fused directly to the amino terminus of the desired hNE inhibitor, under control of the upstream inducible P. pastoris aox1 gene promoter and the downstream aox1 transcription termination and polyadenylation sequences. The expression plasmids were linearized by SacI digestion and the linear DNA was incorporated by homologous recombination into the genome of the P. pastoris strain GS115 by spheroplast transformation, selection for growth in the absence of added histidine and screening for methanol utilization phenotype, secretion levels and gene dose (estimated by Southern Blot). Strains estimated to have about four copies of the expression plasmid integrated as a tandem array into the aox1 gene locus were thus selected.
Cultures of selected strains were first grown in batch mode with glycerol as the carbon source, then, following exhaustion of glycerol, grown in glycerol-limited feed mode to further increase cell mass and derepress the aox1 promoter and finally in methanol-limited feed mode. During the latter phase the aox1 promoter is fully active and the protein is secreted into the culture medium.
The EPI-hNE protein is then purified. The specific purification procedure varies with the specific properties of each protein. Briefly, the culture medium is centrifuged, the supernatant is subjected to microfiltration and subsequently to ultrafiltration, optionally to diafiltration, and then the protein is recovered by ammonium sulfate precipitation and ion exchange chromatography.
European Patent Application No. 00203049.2 and international patent application PCT/FR 01/02699 claiming priority thereof, filed by the applicant company, describe an improved process for the purification of an EPI-HNE protein of pharmaceutical quality, from the culture medium of a host strain for the expression of said proteins, comprising the steps of:
(a) passing a derived part of the culture medium over an expanded, bed of cationic exchange adsorbent in order to recover an eluate,
(b) conducting separation of proteins, according to their hydrophobicity, on the resulting eluate,
(c) passing the resulting eluate over a cationic exchange column,
(d) optionally filtering the resulting medium under sterile conditions, and
(e) optionally lyophilising the resulting filtrate in order to recover an EPI-HNE protein.
The solution obtained at the end of step (d) or a freeze dried powder obtained therefrom can be used to prepare the suspension according to the present invention, said suspension being capable of being incorporated in an inhalable pharmaceutical formulation according to the invention.
The applicant company, having perfected a purification method of EPI-hNE proteins, particularly EPI-hNE-4, has subsequently devoted a great deal of research and effort to the development of pharmaceutical compositions containing the purified EPI-hNE proteins.
In fact, the Applicant Company has concentrated on the development of a buccal inhalable pharmaceutical composition containing a solution of Epi-hNE-4. However, in the course of product development, it was found that the solution of EPI-hNE-4, once in the nebulizer, was unstable and tended to precipitate, rendering the solution turbid.
It was first thought that the precipitated form of the protein would be therapeutically inactive. However, in a surprising and unexpected manner, it was found that the precipitated form was a crystallized form of EPI-HNE4 and that this crystallization did not adversely affect the activity of the protein. The term xe2x80x9ccrystallized form of EPI-hNE4xe2x80x9d here means an insoluble form of this protein, having a rod-like structure and a particle size mainly below 10 xcexcm.
The applicant company thus turned its efforts towards developing a suspension of the EPI-hNE proteins in which the protein would be in crystalline form, said suspension being capable of being incorporated into a pharmaceutical composition, in particular an inhalable pharmaceutical formulation.
It was surprisingly found that it was possible to prepare a suspension which is stable at room temperature under certain specific conditions of concentration and pH, thereby allowing the preparation of pharmaceutical compositions incorporating said suspension which are stable at room temperature. This room temperature stability is of particular importance for ambulatory treatments.
The use of a suspension of EPI-hNE proteins, instead of a solution, constitutes a major advantage in the preparation of inhalable pharmaceutical compositions, insofar as it allows the development of a formulation which is more concentrated in active substance, thereby permitting administration of the drug in a shorter time frame.
This is an important factor in the administration of inhalable drugs since the time period of inhalation required can often be long, which is perceived as a major constraint and may hence lead to poor patient compliance.
Thus, the present invention concerns a suspension of an EPI-hNE protein, said suspension being characterized in that the EPI-hNE protein is present in the form of crystalline particles mostly having a particle size comprised between 1 and 6 xcexcm, in particular between 3 and 6 xcexcm, as determined by laser granulometry, the concentration of the suspension in the EPI-hNE protein being comprised between 1 and 80 mg/ml, preferably between 2 and 50 mg/ml, most preferably depending on the amount of the EPI-hNE protein required for the treated therapeutic condition, in an aqueous vehicle at a pH comprised between 3and 8, preferably 4 and 6, most preferably at a pH of 4.0 to 5.0.
The above suspension is stable as to its biological activity and particle size distribution for a period of at least two months at room temperature.
The aqueous vehicle is preferably a saline solution having an iso-osmotic pressure.
The saline solution may comprise sodium acetate and sodium chloride or sodium citrate.
The EPI-hNE protein is suitably selected from the group consisting of EPI-hNE-1, EPI-hNE-2, EPI-hNE-3, and EPI-hNE-4, preferably EPI-hNE-3 or EPI-HNE4. Most preferably it is EPI-hNE4.
The above suspension then preferably comprises at least 65% of the crystalline particles of EPI-HNE4 having a particle size between 3 and 6 xcexcm, as determined by laser granulometry.
The inhaled mass of this suspension, determined on a Pari LC-star Nebuliser, is about 40 to 50%, which is a good value for an inhalable pharmaceutical formulation.
The MMAD (Median Mass Aerodynamic Diameter) of the nebulisate droplets containing EPI-HNE4 crystallized particles is about 2 xcexcm, as determined by impactor granulometry. This MMAD value is well suited to ensure a high respirable fraction and an effective penetration into the lungs.
A dry powder can be derived from said suspension e.g. by submitting the latter to centrifugation, preferably after adjusting its pH to 3.5 to 4.5, separating the supernatant, gently vacuum-drying the pellet, then homogenizing so as to individualize the particles from the agglomerates. Under these conditions, spheroid-like particles which mostly have a particle size or diameter between 1 and 6 xcexcm, as determined by direct microscopy, are obtained. The dry powder has a size distribution comparable to that of the suspension, when determined in solution by laser granulometry.
A dry powder can also be derived from a solution of EPI-hNE protein, or a freeze-dried powder of EPI-hNE protein.
The invention thus also relates to a dry powder which comprises spheroid-like particles of EPI-hNE-4 mostly having a particle size or diameter of between 1 and 6 xcexcm, preferably at least 75% of the particles having a particle size or diameter between 1 and 3 xcexcm, as determined by direct microscopy, and mostly having a particle size or diameter of between 1 and 6 xcexcm, preferably at least 60% of the particles having a particle size or diameter between 1 and 3 xcexcm, as determined by laser granulometry. This dry powder is suitably obtained by a method comprising the steps of separating, in the above-defined suspension of EPI-hNE protein, the crystals from the liquid phase, discarding the residual water and homogenizing the crumbly compact cake obtained.
The invention also relates to a dry powder comprising spheroid-like particles of EPI-hNE-4, at least 90% of the particles having a particle size or diameter between 0.5 and 4.0 xcexcm, the MMAD being about 2.1 xcexcm, as determined by laser granulometry. This MMAD value is well suited to ensure, when the powder is dispersed in a suitable propellant vehicle, a high respirable fraction and an effective penetration into the lungs. This dry powder may conveniently be obtained by a method comprising the step of spray-drying a solution of EPI-hNE protein.
The invention also concerns an inhalable pharmaceutical formulation comprising the above-defined suspension of the EPI-hNE-4 protein or a dry powder aerosol comprising the above-defined dry powder in a suitable propellant vehicle.
The invention further concerns methods for the preparation of a suspension of EPI-hNE protein, starting either from a solution of EPI-hNE protein or from a freeze-dried powder.
When starting from a solution of EPI-hNE protein, a suitable method for preparing the suspension comprises the steps of:
(a) bringing the pH of the solution to a value comprised between 3.5 and 4.5, so as to allow crystallization of the EPI-hNE protein, and
(b) bringing the pH to a value between 3.0 and 8.0.
The above steps (a) and (b) can suitably be performed at a temperature from 1 to 40xc2x0 C., preferably from 4 to 30xc2x0 C.
When starting from a freeze-dried powder, a suitable method for preparing the suspension comprises the steps of:
(a) solubilizing the EPI-hNE protein in a buffer having a pH below 3.0
(b) bringing the pH of the solution to a value comprised between 3.5 and 4.5, so as to allow crystallization of the EPI-hNE protein, and
(c) bringing the pH to a value comprised between 3.0 and 8.0.
The above steps (a), (b) and (c) can suitably be performed at a temperature from 1 to 40xc2x0 C., preferably from 4 to 30xc2x0 C.
The invention also concerns methods of preparing a dry powder as defined above starting from the above suspension of EPI-hNE protein, a solution or a freeze-dried powder thereof.
When starting from a suspension of EPI-hNE protein the method for preparing the dry powder comprises the steps of separating, in the above suspension, the crystals from the liquid phase, e.g. by centrifugation or filtering on a submicronic filter, discarding the residual water, suitably using a method not causing extensive compacting of the solid phase cake, e.g. by gentle evaporation at a temperature below 40xc2x0 C. and a pressure slightly below atmospheric pressure, and homogenizing the solid cake obtained so as to individualize the agglomerated particles, suitably by techniques well known in the art of milling e.g. using a Pulverisette 5 (planetary mill) from Fritsch or an electric milling apparatus Moulin JK from Laboratoire Moderne, Paris.
When starting from a solution of EPI-hNE protein a suitable method for preparing the dry powder comprises the steps of
(a) bringing the pH of the solution to a value comprised between 3.5 and 4.5, so as to allow crystallization of the EPI-HNE protein.
(b) separating, in the above suspension, the crystals from the liquid phase, e.g. by centrifugation or filtering on a submicronic filter,
(c) discarding the residual water, suitably using a method not causing extensive compacting of the solid phase cake, preferably by gentle evaporation at a temperature below 40xc2x0 C. and a pressure slightly below atmospheric pressure,
(d) homogenizing the solid cake obtained so as to individualize the agglomerated particles, suitably by techniques well known in the art of milling.
The above steps (a) and (b) can suitably be performed at a temperature from 1 to 40xc2x0 C., preferably from 4 to 30xc2x0 C.
When starting from a solution of EPI-hNE protein a preferred method comprises the step of spray-drying this solution. The parameters of this step may conveniently be adjusted to ensure a particle size distribution such as to ensure, when the powder is dispersed in a suitable propellant vehicle, a high respirable fraction and an effective penetration into the lungs.
When starting from a freeze-dried powder of EPI-hNE protein a suitable method for preparing the dry powder comprises the steps of:
(a) solubilizing the EPI-hNE protein in a buffer having a pH below 3.0
(b) bringing the pH of the solution to a value comprised between 3.5 and 4.5, so as to allow crystallization of the EPI-hNE protein.
(c) separating, in the above suspension, the crystals from the liquid phase, preferably by centrifugation or filtering on a submicronic filter.
(d) discarding the residual water, suitably using a method not causing extensive compacting of the solid phase cake, preferably by gentle evaporation at a temperature below 40xc2x0 C. and a pressure slightly below room pressure, and
(e) homogenizing the solid cake obtained so as to individualize the agglomerated particles, suitably by techniques well known in the art of milling.
The above steps (a) and (b) can suitably be performed at a temperature from 1 to 40xc2x0 C., preferably from 4 to 30xc2x0 C.
The invention also concerns the use of the above suspension or dry powder aerosol of an EPI-hNE protein for the preparation of medicaments for treating a disease condition which is due to an excessive activity of hNE.
The disease condition may be, in particular, any respiratory disorder or may be selected from the group consisting of cystic fibrosis, emphysema, ARDS (Acute Respiratory Distress Syndrome) and COPD (Chronic Obstructive Pulmonary Disease).
The examples which follow will serve to better describe the invention, but are in no way to be considered as being limitative.